oasis prime hlb - introducing a new sorbent for the sample ... · from milk (60 compounds in 9 drug...

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Today’s Presenter

Jeremy Shia, Ph.D.

Marketing Manager

Waters Corporation, Milford, MA

Jeremy is currently the Food & Environment marketing manager at Consumables Business Unit of Water Corporation. Previously, Jeremy was a Senior Applications Chemist in our Chemistry Applications Technology group responsible for sample prep development for food testing and food safety. He has published multiple application notes using the QuEChERS method. He has worked in a variety of areas ranging from carbohydrate and vitamin analysis, to multi-residue analysis of veterinary drugs, pesticides and mycotoxins in various food matrices.


Oasis® PRiME HLB - Introducing A New Sorbent

for the Sample Cleanup of Food Matrices

Jeremy Shia, Marketing Manager

Meet The Experts - Webinar

November 19, 2015


Agenda

Why Food Safety Testing is Important

Considerations for sample preparation

Oasis PRiME HLB Introduction

Application of Oasis PRiME HLB for Cleanup of Meat and Milk

Extracts Prior to Multiresidue Veterinary Drug LC-MS/MS

Analysis

Summary


Drivers for Food Safety Testing

Regulations – Surveillance, national monitoring programs, import/export

Economic – Brand differentiation/marketing

– Increased consumer prosperity, greater choice, greater competition

– Green lobby – organic & sustainability

Public Scrutiny / Media Coverage – Brand protection

– Food scares – BSE, dioxins, antibiotics, mycotoxins

– Adulteration/fraud

o wine, orange juice, olive oil, melamine


Substances Which Pose Risk

Environmental contaminants – dioxins, PCBs, metals

Natural toxic substances – mycotoxins, phycotoxins, plant toxins

Pesticide residues

Veterinary drug residues

Contamination in field:

Contamination while processing:

Processing contaminants

Additives – illegal dyes

Packaging migration

Authenticity/traceability (GMO)

Deliberate adulteration


Scientific Challenges in Food Safety Testing

Sensitivity is required to accurately identify contaminants in a wide variety sample matrices – Regulated methods

– Emerging contaminants

High throughput is a necessity – Hundreds of samples

– Fast turnaround time

Method ruggedness and reliability is essential – Co-eluting endogenous materials can result in reduced assay accuracy

Data quality must be maintained – Better, more informed decisions


Why Do Sample Preparation?

Need to remove matrix interferences

– Increase signal to noise by simplifying the chromatographic separation

o Enables better, more consistent quantitation

– Reduce variability in analytical results due to matrix inconsistencies

o Higher, more consistent recovery

o Minimize matrix effects

o Less rework

– Increase column lifetime

o Fewer columns need to be replaced

– Reduces system downtime

o Less time spent with wrenches or waiting for service

Need to concentrate analyte of interest

– Present in low levels = difficult to quantitate

Need to transfer (extract) analytes of interest into a solution that can be tested


Sample Preparation

How can I turn this?

Into this?


Sample Preparation Techniques: Which One?

How do you choose a technique to clean up complex sample

matrices?

– Filtration / Dilution

– Protein precipitation (PPT)

– Liquid-liquid extraction (LLE)

– QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe)

– Solid-phase extraction (SPE)

Objectives: Simplest technique

Fastest preparation procedure Cleanest extracts

“Good Enough” Sample Preparation


Method Requirements: Multi-residue vs. Compound-specific analysis

Multi-Residue/ Multi-Class

Compound or Class Specific

Entire procedure (sample prep & analytical method)

Generic to a diverse set of analytes

Specific for one compound or class of compounds

Sample Preparation Protocol

Simple (one or two steps) Multi-step

Goal of Sample Cleanup

Speed

Recovery & cleanup may be compromised for a large

number of analytes

Maximizing recovery & matrix cleanup

Minimizing interference/ion

suppression

Level of Sample Cleanup Minimum/moderate Maximum

Detection Techniques Tandem MS, Time-of-Flight

(LC-MS and GC*-MS) Single quad MS, UV, FLR,

ELS, GC* (FID or MS)

*GC typically requires a higher level of sample cleanup even for multi-residue analyses


Introducing Oasis PRiME HLB


What is Oasis PRiME HLB?

PROCESS

ROBUSTNESS

improvements in…

MATRIX EFFECTS

EASE of USE

What’s in a name (what does PRiME mean)?


Oasis PRiME HLB in 3 Words

SIMPLER

FASTER

CLEANER


Oasis PRiME HLB – What is it?

A reversed-phase solid phase extraction device

– PATENT PENDING

Designed to simplify solid phase extraction

– SIMPLER

o Easy protocols that result in cleaner samples

o No condition and equilibration steps are required

o Easy to implement into laboratory workflows without SPE

expertise


SIMPLER – 3 Step Catch and Release Protocol

No SPE expertise required

Reversed-phase

retention for

aqueous samples

Suitable for compound/class

specific analysis

Load:

Pre-Treated Sample

Wash:

5% MeOH

Elute:

90/10

Acetonitrile/MeOH Wash and elution

steps can be adjusted if desired


3-Step Protocol Example: Test Analyte Properties

Name pKa Log P Comments

1B Azidothymidine (AZT) 9.68 0.05 Antiretroviral drug for HIV/AIDS

2B 7-Hydroxycoumarin 7.8 1.6 Gradient in sunscreen, absorb UV

3A Phenacetin 2.2 1.6 Pain, fever reducer

4N Betamethasone -- 1.1 Anti-inflammatory and

immunosuppressive

5B Protriptyline 8.2 4.4 Antidepressant

6A Alprazolam 2.4 4.9 Panic and anxiety disorders

7B Amitriptyline 9.7 4.8 Antidepressant

8A Naproxen 4.2 3.2 Pain, fever reducer

9B Propranolol 9.5 2.5 Hypertension

Drug Panel Mixture: Highly variable hydrophobicities, wide pKa

range and Log Ps


1 2

3

4 7

8

9

3-Step Protocol Example: Chromatogram

1. AZT (Azidothymidine)

2. Propranolol

3. 7-Hydroxycoumarin

4. Phenacetin

5. Protriptyline

6. Amitriptyline

7. Betamethasone

8. Alprazolam

9. Naproxen

5 6


3-Step Protocol Example: Recovery and Matrix Effects

-20

0

20

40

60

80

100

120

Luckycat 1cc plasma 500-500 Luckycat 1cc MERecovery Matrix Effects

3-Step Protocol CONCLUSION HIGH analyte recoveries (>80%) and

LOW (<15%) matrix effects


3-Step Protocol Example: Excellent Reproducibility

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Batch #1 Batch #2 Batch #3 Batch #4 Batch #5

3-Step Protocol CONCLUSION Reproducible recoveries for all acidic, basic and neutral

compounds batch to batch, with an average recovery of 87%











Speed


number of analytes



suppression





√


SIMPLER – 2 Step Pass-Through

2 Step Protocol – Ideal for

high organic (ACN) samples,

like meat or milk extracts

Analytes not retained by the

sorbent

Suitable for multi-class

compounds analysis

i.e. Multi-residue veterinary

drugs analysis

Load – Collect

Interferences Retained

Analytes Pass Through

Rinse – Collect

Analytes Pass Through

Note: Rinse step is optional


Recovery of Multi-residue Veterinary from Milk (60 compounds in 9 drug classes)

One single method replaces 9 separate methods!!! Excellent recoveries ranging from 70% to 120% with precision (RSD) < 20% (n=5) for all compounds (Average recovery 91%, %RSD @ 6 (n=5)) Recovery values are a subject to the initial milk extraction efficiency

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Cim

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Salb

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Clindam

ycin

Ery

thro

mycin

kitasam

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Lin

com

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Spiram

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Tilm

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Tylo

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Sulfachlo

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Sulfaclo

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Sulfadim

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oxin

e

Sulfaguanid

ine

Sulfam

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zin

e

sulfam

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r

Sulfam

eth

azin

e

sulfam

eth

izole

Sulfam

eth

oxypyridazi…

sulfanilaceta

mid

e

sulfaphenazole

Sulfapyridin

e

sulfis

om

idin

e

Trim

eth

oprim

Cin

oxacin

Cip

rofloxacin

Danofloxacin

Diflo

xacin

Enoxacin

Enro

floxacin

Flu

mequin

e

Lom

efloxacin

Marb

ofloxacin

Nalidix

ic a

cid

Norf

loxacin

Ofloxacin

Orb

iflo

xacin

Oxolinic

acid

Pefloxacin

Sara

floxacin

Chlo

ram

phenic

ol

florf

enic

ol

Thia

mphenic

ol

penic

illin V

Beta

meth

asone

Cort

isone

Dexam

eth

asone

Hydro

cort

isone

Mepre

dnis

one

Meth

ylp

rednis

olo

ne

Pre

dnis

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ne

Triam

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olo

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Triam

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…

Cefo

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Ceft

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Speed


number of analytes



suppression





*GC typically requires a higher level of sample cleanup even for multi-residue analyses

√





– SIMPLER

– FASTER

o Faster flows though the device

o More even flows across cartridges and plates with less plugging

o Faster overall processing with the elimination of condition and

equilibration steps combined faster flows (especially important with

cartridges)


FASTER - Flows

Faster, more even sample flows across cartridges and plates

with less plugging

Loading compared to Oasis HLB with 4 inch Hg, N=4

Matrix Device Format Oasis PRiME HLB Speed Increase

1:1 Diluted Plasma µElution Plate 2 - 3X Faster

1:1 Diluted Plasma 1cc / 30mg Cartridge 4X Faster

1:1 Diluted Urine 30 mg Plate 6X Faster

1:1 Diluted Urine 10 mg Plate 2X Faster

1:1 Diluted Milk 3cc / 60 mg Cartridge 1 - 2X Faster

1:1 Diluted Milk 6cc / 200 mg Cartridge 2 - 3X Faster

FASTER flows across multiple devices and sample matrices





– SIMPLER

– FASTER

– EVEN CLEANER

o Removes more than 95% of common matrix interferences, such as

salts, proteins and phospholipids, with the generic 3-step protocol

o Removes at least 90% more phospholipids than the generic

protocol with Oasis HLB


Lipids

Amphipathic, surfactant-like

property

Serve as a major structural

component of most biological

membranes

Form lipid bilayer in cell

membranes of organisms

Example: phosphatidylcholine or

lecithin

– Often found in animal and plant

tissues, such as egg yolks, milk,

soybeans, animal cells

Phospholipids - A Brief Introduction

Phosphate group

“hydrophilic head”

Fatty acids

“hydrophobic tails”


Phospholipid Build Up Can Lead to Matrix Effects and Unpredictable Results

Using protein precipitation, residual phospholipids build up on the column during the processing of one analytical batch (50 samples total) The bottom chromatogram shows the earliest and latest eluting parent compounds superimposed over the phospholipid trace, illustrating co-elution. This may cause matrix effects. Also note the elution of a significant phospholipid peak at the end of the gradient re-equilibration. This may cause matrix effects and a decrease in robustness.

Application Note: Rapid, Reliable Metabolite Ratio Evaluation for MIST Assessments in Drug Discovery and Preclinical Studies, 720004453en

Application Note: Rapid, Reliable Metabolite Ratio Evaluation for MIST Assessments in Drug Discovery and Preclinical Studies, 720004453en Danaceau et al. 2014 Bioanalysis 6(6) 761-771


Oasis PRiME HLB Summary

Oasis PRiME HLB is the next generation SPE device that sets the

new performance standard for routine analyses

– Best choice for samples that contain proteins, fats, and/or lipids and

can be prepared by reversed-phase ‘catch-and-release’ SPE or ‘pass-

through’ SPE

SIMPLER: Streamlined protocols, no condition and equilibration

steps, easy to implement into laboratory workflows without SPE

expertise

FASTER: Faster flows through device, more even flows across

cartridges and plates with less plugging, faster overall

processing

CLEANER: Reduced matrix effects, phospholipid and fat removal

in the pass-through method, less column and/or instrument

contamination


Application of Oasis PRiME HLB for Cleanup

of Meat and Milk Extracts Prior to

Multiresidue Veterinary Drug LC-MS/MS

Analysis


US DA Residue Methods for Veterinary Drug Analysis

Method Analytes Sample Matrix

R04b β-Adrenergic agonists Muscle

R06 β-Lactam Kidney and muscle

R08b Chloramphenicol Muscle

R18 Macrolide Liver, kidney, and muscle

R27d Sulfonamides Liver and muscle

R32 Fluoroquinolone Liver and muscle

R46 Penicillin G Kidney, liver, and muscle

Using Oasis PRiME HLB -Enable multi-class compounds to be analyzed using one sample prep method.

Combined


Challenges Creating a Single Extraction Method

Compound diversity

– Large number of diverse group and classes

– Different physical and chemical properties

o Polarity

o pKa

– Parent drugs and their metabolites

Matrix complexity

– Matrix effects – proteins, fats, phospholipids, salts

– Protein binding

– Low limits of detection vs. co-extracted material – ion suppression

– Chelating - Tetracycline and similar drugs can form bonds with calcium (milk matrix)


Multi-Residue Analysis - Composition of Milk and Meat

Typical Cow’s Milk

– Approximately 14 % solids

o 4 % fat

o 0.3-1 % phospholipid

o 4 % protein

o 5 % sugar (lactose)

o 85 % water

Typical Pork Muscle

—Approximately 30 % solids

o 5-20 % fat

o 1-3 % phospholipid

o 15-25 % protein

o ~70% water

√

√

√

√


Veterinary Drug Classes

tetracycline

Tetracycline Fluoroquinolone

enrofloxacin

Sulfonamide

sulfamethazine

Macrolide

erythromycin

Beta-Lactam

oxacillin

Beta-adrenergic

salbutamol

Steroid

dexamethasone

Amphemicol

Chloramphemicol

LogP 2.05, pKa 3.75

LogP -1.3, pKa -2.2

LogP 0.44, pKa (basic) 9.4

LogP 2.37 , pKa(basic)8.3


Veterinary Drugs Extraction Methods

Typical Sample Preparation Strategies

– Precipitation/extraction with strong buffer (McIlvaine pH 4) followed by SPE

o good for tetracyclines, beta-adrenergics, polar sulfonamides, fair for fluoroquinolones,

o poor recovery of most other compounds

– Precipitation/extraction with 3:1 acidic acetonitrile with SPE cleanup

o excellent protein precipitation

o poor recovery of tetracyclines, beta-adrenergics, polar sulfonamides

o good recovery of most other compounds


Typical %recoveries from milk Comparison of precipitation/extraction techniques

Drug Class 3:1 ACN Aq Buffer 1:1 ACN*

β-adrenergic <10 ~100 >80

Tetracycline <25 >70 >25

Fluoroquinolone >50 >50 >50

Macrolide >60 <35 >60

Beta-Lactam >70 <30 >70

Steroid >70 <10 >70

See also: Stolker et. al., Anal. Bioanal. Chem. 391, 2309 (2008)


Why not QuEChERS for Sample Prep of Veterinary Drugs?

Some classes of veterinary drugs have good recovery using QuEChERS

(i.e., avermectins)

– However, too many have low recovery (< 50%)

First step of veterinary drug extraction is similar

– However, no salts are added for veterinary drug analysis and pass

through SPE is preferred

Pesticides are generally more non-polar and less water-soluble than

veterinary drugs

– At least 80% partition into ACN layer when DisQuE salts are added

In comparison, a larger portion of veterinary drugs partition into the

salt-saturated water layer


Multi-Residues Veterinary Drug Analysis - Sample Extraction

MILK

Protein Precipitation

Add 4 mL of 0.2 % formic acid ACN to

1 mL of sample

Pass-Through SPE

Cleanup

TISSUE

Extraction/Precipitation

Add 10 mL of 0.2 % formic acid in 80:20 ACN/water to

2.5 g of sample

Provides good recovery of

most analytes

Removes proteins sugars

and salts

Waters Application Notes Optimized Extraction and Cleanup Protocols for LC/MS/MS Multi-Residue Determination of Veterinary Drugs in Milk (720004089en) Optimized Extraction and Cleanup Protocols for LC-MS/MS Multi-Residue Determination of Veterinary Drugs in Edible Muscle Tissues (720004144en)

Centrifuge

Take aliquot


Pass-Through SPE *

Cleanup

Removes fats and non-polar pigments

Sep-Pak

C18

Oasis

PRiME HLB

SPE Strategy for Vet Drugs Pass-Through Cleanup*

Removes fats, non-polar pigments and phospholipids

*Also known as “Chemical Filtration”


Oasis PRiME HLB – 1 Step Pass-Through - No Rinse Step, Even More SIMPLER

1 Step Protocol – Ideal for

high organic (ACN) samples,

like meat or milk extracts

Analytes not retained by the

sorbent

Suitable for multi-class

compound analysis

i.e Multi-residue veterinary

drugs analysis

Pass-Thru and Collect

Matrix Interferences

Retained

1-Step Pass-Through


Multi-Residue Veterinary Drugs in Milk and Tissue

Sample pre-treatment (Milk):

To 1mL of milk, add 4 mL of 0.2% formic acid (FA) in acetonitrile (ACN), mix well. Centrifuge for 5 min at 10000 rpm. An aliquot of the supernatant is taken for SPE cleanup.

Sample pre-treatment (Tissue):

Extract 2.5 g tissue with 10 mL of 0.2% formic acid (FA) in 80:20 ACN/Water and mix well. Centrifuge for 5 min at 10000 rpm. An aliquot of the supernatant is taken for SPE cleanup.

Pass-Thru Cleanup:

The vacuum was set to 1-2 psi. Approximately 0.5 mL of the supernatant was passed-through the Oasis PRiME cartridge and collected. A 0.3 mL aliquot of the pass-thru cleanup sample was taken and diluted three-fold with aqueous 10 mM ammonium formate buffer (pH 4.5) prior to UPLC-MS/MS analysis.

* Typically, an exact portion of the pass-thru fraction is evaporated or simply diluted with mobile phase, depending on the instrumental conditions


UPLC Conditions

LC system: ACQUITY UPLC I-Class Column: ACQUITY UPLC CSH™ C18, 1.7µm, 100 mm x 2.1 mm (ID)

Mobile phase: — A: 0.1% formic in water

— B: 0.1% formic acid in acetonitrile Injection volume: 5 μL Injection mode: partial loop injection

Column temperature 30 °C Weak Needle Wash: 10:90 acetonitrile:water (600 μL)

Strong Needle Wash: 50:30:40 water:acetonitrile:IPA (200 μL) Seal wash: 10:90 acetonitrile: water

615850.47.0

69550.43.9

69550.44.9

615850.45.0

40

15

% B

6600.42.5

Initial850.4Initial

Curve% AFlow

(mL/min)

Time

(min)

615850.47.0

69550.43.9

69550.44.9

615850.45.0

40

15

% B

6600.42.5

Initial850.4Initial

Curve% AFlow

(mL/min)

Time

(min)

Gradient


MS Conditions

Mass Spectrometer: Waters Xevo TQ-S

Positive Electrospray (except negative for chloramphenicol)

Source Temperature: 150°C

Desolvation Temperature: 500°C

Desolvation Gas Flow: 1000 L/Hr

Cone Gas Flow: 30 L/Hr

Collision Gas Flow: 0.15 mL/Min

Data Management: MassLynx v4.1


MRM Transitions

Compounds MRM Cone(V) Collision(eV

)

Spike Level (low,high)

µg/kg

Calibration Range µg/kg Corr (R2) RT Compounds MRM Cone(V)

Collision(eV)

Spike Level (low,high)

µg/kg

Calibration Range µg/kg Corr (R2) RT

Amoxicillin 366.2>349.1 30 8 12.5, 50 6.25-100 0.9978 0.7 Oxacillin 402.2>160.0 30 12 25, 100 12.5-200 0.9974 1.06

366.2>114.0 30 20 402.2>243.1 30 15

Carbadox 263.0>231.0 25 15 25, 100 12.5-200 0.9978 1.43 Oxytetracycline 461.2>426.2 30 21 25, 100 12.5-200 0.9952 1.06

263.0>145.0 25 20 461.2>443.1 30

Ceftiofur 524.3>241.1 30 16 250, 1000 125-2000 0.9975 2.84 Penicillin 335.2>160.1 20 30 12.5, 50 6.25-100 0.9903 3.46

524.3>285.0 30 16 335.2>176.1 20 30

Chloramphenicol 321.0>152.1 30 17 25, 100 12.5-200 0.9943 1.64 Phenylbutazone 309.4>160.0 37 20 25, 100 12.5-200 0.9915 4.29

321.0>257.1 30 15 309.4>103.9 37 20

Chlortetracycline 479.3>444.2 30 21 25, 100 12.5-200 0.9955 0.97 Ractopamine 302.2.164.1 30 15 75, 300 37.5-600 0.9915 1.03

479.3>462.2 30 18 302.2>107.0 30 27

Ciprofloxacin 332.1>288.1 30 18 25, 100 12.5-200 0.9918 2.99 Salbutamol 240.2>148.1 30 20 25, 100 12.5-200 0.9907 0.61

332.1>231.1 30 40 240.2>222.1 30 12

Cortisol 363.2>121.0 42 52 50, 200 25-400 0.9989 3.45 Sulfamerazine 265.0>92.0 30 28 25, 100 12.5-200 0.9918 0.91

363.2>91.03 30 22 265.0>156.0 30 15

Dexamethasone 393.2>373.2 30 10 25, 100 12.5-200 0.998 1.09 Sulfamethazine 279.1>186.0 30 16 25, 100 12.5-200 0.9971 1.56

393.2>355.3 30 15 279.1>92.0 30 28

Enrofloxacin 360.4>245.0 50 25 50, 200 25-400 0.9961 2.26 Sulfanilamide 156.0>92.0 30 15 25, 100 12.5-200 0.9977 1.73

360.4>316.1 50 25 156.0>65.0 30 25

Erythromycin 734.4>158.1 30 32 2.5, 10 1.25-20 0.9982 0.61 Tetracycline 445.3>154.0 30 26 25, 100 12.5-200 0.997 1.15

734.4>576.5 30 20 445.3.410.2 30 21

Lincomycin 407.2>126.1 36 34 12.5, 50 6.25-100 0.9931 1.03 Tylosin 916.5>174.1 57 40 5, 20 2.5-40 0.9938 2.48

407.2>359.3 36 20 916.5>101.1 57 45

Lomefoxacin 352.1>265.1 31 22 50, 200 25-400 0.996 3.79

352.1>308.1 31 16


Recovery of Multi-residue Veterinary from Milk

Multi-residue veterinary drugs in milk including 60 compounds in 9 drug classes compounds Excellent recoveries were obtained in the range of 70% to 120% with precision (RSD) < 20% (n=5) for all compounds (Average recovery 91%, %RSD @ 6 (n=5))

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80

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140

Cim

ate

rol

Cle

nbute

rol

Racto

pam

ine

Salb

uta

mol

Terb

uta

line

Tulo

bute

rol

Zilpate

rol

Clindam

ycin

Ery

thro

mycin

kitasam

ycin

Lin

com

ycin

Spiram

ycin

Tilm

icosin

Tylo

sin

Sulfachlo

rpyridazin

e

Sulfaclo

zin

e

Sulfadim

eth

oxin

e

Sulfaguanid

ine

Sulfam

era

zin

e

sulfam

ete

r

Sulfam

eth

azin

e

sulfam

eth

izole

Sulfam

eth

oxypyridazi…

sulfanilaceta

mid

e

sulfaphenazole

Sulfapyridin

e

sulfis

om

idin

e

Trim

eth

oprim

Cin

oxacin

Cip

rofloxacin

Danofloxacin

Diflo

xacin

Enoxacin

Enro

floxacin

Flu

mequin

e

Lom

efloxacin

Marb

ofloxacin

Nalidix

ic a

cid

Norf

loxacin

Ofloxacin

Orb

iflo

xacin

Oxolinic

acid

Pefloxacin

Sara

floxacin

Chlo

ram

phenic

ol

florf

enic

ol

Thia

mphenic

ol

penic

illin V

Beta

meth

asone

Cort

isone

Dexam

eth

asone

Hydro

cort

isone

Mepre

dnis

one

Meth

ylp

rednis

olo

ne

Pre

dnis

olo

ne

Triam

cin

olo

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Triam

cin

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…

Cefo

taxim

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Ceft

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cephapirin


Phospholipids Removal From Milk

Milk sample processed by protein precipitation only

Precipitated milk sample cleaned up with

Oasis PRiME HLB

Oasis PRiME HLB removed many more phospholipids compared to protein precipitation


Phospholipid Removal From Shrimp Extract


Recovery of Veterinary Drugs from Salmon Tissue (9 Classes of Drugs)

Total Method Recovery

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Recovery

Recovery from Salmon


Recovery of Veterinary Drugs from Shrimp Tissue (9 Classes of Drugs)

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Reco

very

Recovery from Shrimp Total Method Recovery


Recovery of Veterinary Drugs Discussion

Method recovery averages ~ 80% with range from 50-103%

– Most of any recovery loss occurs in the initial extraction/precipitation

step

o Not all classes of compounds are extracted equally

• Penicillins > 70%

• Sulfonamides > 85%

• Fluroquinolones> 75%

• Tetracyclines > 40%

What recovery losses are caused by the pass-thru cleanup?


Oasis PRiME HLB Pass-Thru Cleanup Recovery

Salmon matrix blank extracts were spiked with the veterinary drugs and then cleaned up by passing through the Oasis PRiME HLB cartridge. This experiment eliminates the recovery loss contribution from the initial protein precipitation/extraction . This salmon data presented below shows the pass-thru cleanup efficiency versus the total method recovery. Shrimp data were similar.

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Recovery

Recovery from Salmon

Method

Recovery

Pass-Thru Recovery


Options for Compounds Like Tetracycline

Option A

– Based on the method validation result, build in concentration factor when

considering the reporting levels. Perform confirmation test if needed.

Option B

– Exclude tetracycline from the list of this screening method. Analyze

tetracyclines using individual analytical method –

o Tetracyclines are strong chlelating agents – EDTA or oxalic acid is

added to the McIlvaine buffer to sequester calcium

o Follow by Oasis MAX cleanup

Option C

– Extract sample again using the optimized method for tetracycline, and

combine the extracts together. After adjusting the solvent by adding

ACN, then pass through Oasis PRiME HLB.


Summary

Worldwide regulatory requirements are expanding to include

more compounds

– 400+ pesticides and 100+ veterinary drugs

– Need for good multi-residue screening methods are required to meet

these regulations

– Need maximum number of compounds in a minimum number of

methods for multi-residue screening

PRiME HLB operated as “chemical filter”. Interference such as

fatty matrix and phospholipids are retained by the sorbent, and

analytes are allowed to be passed through.

Oasis PRiME HLB effectively removes majority of fatty matrix

and phospholipids from milk and tissues, and gives the cleanest

possible sample extracts for multi-residue veterinary drug

analysis.


Questions?


Thank You for Attending!

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oasis prime hlb - introducing a new sorbent for the sample ... · from milk (60 compounds in 9 drug...

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